Granulocyte colony-stimulating factor (G-CSF) is a 20 kilodalton (kDa) glycoprotein that promotes the proliferation of progenitor cells and induces their differentiation into neutrophils. In addition, G-CSF prolongs the survival of mature neutrophils and activates their functions Human G-CSF (hG-CSF) is produced by monocytes, macrophages, fibroblasts and endothelial cells (see, for example, Moore, Annu. Rev. Immunol., 9:159-191, 1991; Nicola, Annu. Rev. Biochem., 58:45-77, 1991). The biological effects of G-CSF are mediated through its interaction with the G-CSF receptor (G-CSF-Rc) expressed on the surface of bone marrow hematopoietic progenitors and cells of the myeloid lineage. Upon binding G-CSF, the receptor is activated and undergoes homodimerization, followed by phosphorylation of Janus family of tyrosine kinases. Subsequently, a series of intracellular signal transduction events take place, leading to the increase of the number of progenitor cells, their maturation into neutrophils, and further activation of effector functions in mature neutrophils (see, for example, Demetri et al., Blood, 78:2791-2808, 1991). Therefore, G-CSF plays an essential role not only in the regulation and maintenance of hematopoiesis, but also in host defense against infection and inflammation.
Recombinant human G-CSF (rhG-CSF) is widely used in the treatment of patients with neutropenia as a result of receiving chemotherapy. Administration of rhG-CSF is effective in restoring functioning neutrophils to these patients, leading to a decrease of infection-related events. Use of rhG-CSF allows intensified dosing or scheduling of chemotherapeutic agents that may be of benefit to cancer patients. Besides chemotherapy-induced neutropenia, rhG-CSF has been used for the treatment of myelosuppression after bone marrow transplantation, acute leukemia, aplastic anemia, myelodysplastic syndrome, severe chronic neutropenias, and mobilization of peripheral blood progenitor cells for transplantation (see, for example, Welte et al., Blood, 88:1907-1929, 1996).
The elimination half-life of the serum concentration of rhG-CSF is approximately 3 to 4 h for intravenous or subcutaneous administration. The safety profile and patient tolerance of rhG-CSF are good with medullary bone pain being the only frequent and significant side effect. The relatively low toxicity of rhG-CSF has made it feasible to develop longer-acting derivatives to decrease the inconvenience of the daily or twice-daily injection schedule. Attachment of polyethylene glycol (PEG) to various proteins, including G-CSF, has been reported to yield derivatives with higher in vivo potency due to their longer half-lives (see, for example, Zalipsky et al., in “PEG chemistry: biotechnical and biomedical applications”, pp. 347-370, 1992). PEG-conjugated proteins usually have considerably lower in vitro biological activity than their unmodified parent proteins (Eliason et al., Stem Cells, 18:40-45, 2000). The increased in vivo potency of these modified proteins is, at least in part, due to decreased removal by the kidney in a manner proportional to their molecular weight (Yamaoda et al., J. Pharmaceut. Sci., 83:601-606, 1994). We unexpectedly discover that it is possible to increase the potency of hG-CSF through prolonging its half-life as well as enhancing its biological activity is to attach the Fc region derived from human IgG at the C-terminus of hG-CSF, as described in this invention.
Immunoglobulins of IgG class are among the most abundant proteins in human blood. Their circulation half-lives can reach as long as 21 days. Fusion proteins have been reported to combine the Fc regions of IgG with the domains of another protein, such as various cytokines and soluble receptors (see, for example, Capon et al., Nature, 337:525-531, 1989; Chamow et al., Trends Biotechnol., 14:52-60, 1996); U.S. Pat. Nos. 5,116,964 and 5,541,087). The prototype fusion protein is a homodimeric protein linked through cysteine residues in the hinge region of IgG Fc, resulting in a molecule similar to an IgG molecule without the CH1 domains and light chains. Due to the structural homology, Fc fusion proteins exhibit in vivo pharmacokinetic profile comparable to that of human IgG with a similar isotype. This approach has been applied to several therapeutically important cytokines, such as IL-2 and IFN-α2a, and soluble receptors, such as TNF-Rc and IL-5-Rc (see, for example, U.S. Pat. Nos. 5,349,053 and 6,224,867). It is desirable to extend the circulating half-life of G-CSF and/or to increase its biological activity by making fusion proteins containing G-CSF linked to the Fc portion of the human IgG protein as disclosed and/or described in this invention.
Erythropoietin (EPO) derivatives, such as dimers, have been reported. Relative to the EPO monomer, a fusion protein consisting of two complete EPO domains separated by a 3- to 7-amino acid peptide linker exhibited reduced activity (Qiu et al., J. Biol. Chem., 273:11173-11176, 1998). However, when the peptide linker between the two EPO domains was 17 amino acids in length, the dimeric EPO molecule exhibited considerably enhanced in vitro and in vivo activities (see, for example, Sytkowski et al., J. Biol. Chem., 274:24773-24778, 1999; U.S. Pat. No. 6,187,564). The length of the peptide linker between the two hematopoietic growth factors is important, while not bound by this theory, presumably due to its effect on the flexibility of such molecular forms. We find that this approach is generally applicable to other therapeutic proteins, including G-CSF. We'll also refer this to this as a flexible peptide linker.
In most of the reported Fc fusion protein molecules, a hinge region serves as a spacer between the Fc region and the cytokine or soluble receptor at the amino-terminus, allowing these two parts of the molecule to function separately (see, for example, Ashkenazi et al., Current Opinion in Immunology, 9:195-200, 1997). A human G-CSF fusion protein with an appropriate peptide linker between the hG-CSF and Fc moieties (hG-CSF-L-Fc) is more active than rhG-CSF, with in vitro activity at least 2-fold as that of rhG-CSF on a molar basis. It is discovered according to this invention that an added peptide linker present between hG-CSF and a human IgG Fc variant enhances the in vitro biological activity of the hG-CSF-L-Fc molecule in two ways: (1) keeping the Fc region away from the G-CSF-Rc binding sites on G-CSF, and (2) keeping one G-CSF from the other G-CSF domain, so both G-CSF domains can interact with G-CSF-Rc on the granulocyte procursor cells independently. For the present invention, a flexible peptide linker of about 20 or fewer amino acids in length is preferred. More preferably, the peptide linker should have at least two amino acids in length. Furthermore, it is even more preferable to use a peptide linker comprising two or more of the following amino acids: glycine, serine, alanine, and threonine.
The Fc region of human immunoglobulins plays a significant role in immune defense for the elimination of pathogens. Effector functions of IgG are mediated by the Fc region through two major mechanisms: (1) binding to the cell surface Fc receptors (FcγRs) can lead to ingestion of pathogens by phagocytosis or lysis by killer cells via the antibody-dependent cellular cytotoxicity (ADCC) pathway, or (2) binding to the C1q part of the first complement component C1 initiates the complement-dependent cytotoxicity (CDC) pathway, resulting in the lysis of pathogens. Among the four human IgG isotypes, IgG1 and IgG3 are effective in binding to FcγR. The binding affinity of IgG4 to FcγR is an order of magnitude lower than that of IgG1 or IgG3, while binding of IgG2 to FcγR is below detection. Human IgG1 and IgG3 are also effective in binding to C1q and activating the complement cascade. Human IgG2 fixes complement poorly, and IgG4 appears quite deficient in the ability to activate the complement cascade (see, for example, Jefferis et al., Immunol. Rev., 163:59-76, 1998). For therapeutic use in humans, it is essential that when hG-CSF-L-Fc binds to G-CSF-Rc on the surface of the progenitor cells or other cells of the myeloid lineage, the Fc region of the fusion protein will not mediate undesirable effector functions, leading to the lysis or removal of these cells. Accordingly, the Fc region of hG-CSF-L-Fc must be of a non-lytic nature, i.e. the Fc region must be inert in terms of binding to FcγRs and C1q for the triggering of effector functions. It is clear that none of the naturally occurring IgG isotypes is suitable for use to produce the hG-CSF-L-Fc fusion protein. To obtain a non-lytic Fc, certain amino acids of the natural Fc region have to be mutated for the attenuation of the effector functions.
By comparing amino acid sequences of human and murine IgG isotypes, a portion of Fc near the N-terminal end of the CH2 domain is implicated to play a role in the binding of IgG Fc to FcγRs. The importance of a motif at positions 234 to 237 has been demonstrated using genetically engineered antibodies (see, for example, Duncan et al., Nature, 332:563-564, 1988). The numbering of the amino acid residues is according to the EU index as described in Kabat et al. (in Sequences of Proteins of Immunological Interest, 5th Edition, United States Department of Health and Human Services, 1991). Among the four human IgG isotypes, IgG1 and IgG3 bind FcγRs the best and share the sequence Leu234-Leu-Gly-Gly237 (only IgG1 is shown in FIG. 1). In IgG4, which binds FcγRs with a lower affinity, this sequence contains a single amino acid substitution, Phe for Leu at position 234. In IgG2, which does not bind FcγRs, there are two substitutions and a deletion leading to Val234-Ala-Gly237 (FIG. 1). To minimize the binding of Fc to FcγR and hence the ADCC activity, Leu235 in IgG4 has been replaced by Ala (see, for example, Hutchins et al., Proc. Natl. Acad. Sci. USA, 92:11980-11984, 1995). IgG1 has been altered in this motif by replacing Glu233-Leu-Leu235 with Pro233-Val-Ala235, which is the sequence from IgG2. This substitution resulted in an IgG1 variant devoid of FcγR-mediated ability to deplete target cells in mice (see, for example, Isaacs et al., J. Immunol., 161: 3862-3869, 1998).
A second portion that appears to be important for both FcγR and C1q binding is located near the carboxyl-terminal end of CH2 domain of human IgG (see, for example, Duncan et al., Nature, 332:738-740, 1988). Among the four human IgG isotypes, there is only one site within this portion that shows substitutions: Ser330 and Ser331 in IgG4 replacing Ala330 and Pro331 present in IgG1, IgG2, and IgG3 (FIG. 1). The presence of Ser330 does not affect the binding to FcγR or C1q. The replacement of Pro331 in IgG1 by Ser virtually abolished IgG1 ability to C1q binding, while the replacement of Ser331 by Pro partially restored the complement fixation activity of IgG4 (see, for example, Tao et al., J. Exp. Med., 178:661-667, 1993; Xu et al., J. Biol. Chem., 269:3469-3474, 1994).
We discover that at least three Fc variants (vFc) can be designed and/or used for the production of hG-CSF-L-vFc fusion proteins (FIG. 1). Human IgG2 Fc does not bind FcγR but showed weak complement activity. An Fcγ2 variant with Pro331Ser mutation should have less complement activity than natural Fcγ2 while remain as a non-binder to FcγR. IgG4 Fc is deficient in activating the complement cascade, and its binding affinity to FcγR is about an order of magnitude lower than that of the most active isotype, IgG1. An Fcγ4 variant with Leu235Ala mutation should exhibit minimal effector functions as compared to the natural Fcγ4. The Fcγ1 variant with Leu234Val, Leu235Ala and Pro331Ser mutations also will exhibit much less effector functions than the natural Fcγ1. These Fc variants are more suitable for the preparation of the G-CSF fusion proteins than naturally occurring human IgG Fc. It is possible that other replacements can be introduced for the preparation of a non-lytic Fc without compromising the circulating half-life or causing any undesirable conformational changes.
There are many advantages with the present invention. The increased activity and prolonged presence of the hG-CSF-L-vFc fusion protein in the serum can lead to lower dosages as well as less frequent injections. Less fluctuations of the drug in serum concentrations also means improved safety and tolerability. Less frequent injections may result in better patient compliance and quality of life. The hG-CSF-L-vFc fusion protein containing a non-lytic Fc variant will therefore contribute significantly to the management of a variety of conditions associated with an impaired immune or hematopoietic system, including cancer chemotherapy, leukemias, anemias AIDS, bone marrow transplantation, and chronic neutropenias.